Epstein Barr Virus (EBV)-associated lymphoma remains a major problem among AIDS patients, and Kaposis sarcoma, which has declined in frequency in recent years, can still cause debilitating disease in HIV-infected patients. In collaboration with Peter Burbelo, we are examining antibody responses to EBV and HHV-8 in patients who develop either lymphoma (EBV) or Kaposis sarcoma (HHV-8), using a new technology developed by Dr. Burbelo, the luciferase immunoprecipitation system (LIPS), which allows rapid detection of antibodies over a broad dynamic range. For HHV-8, our goal is to see if we can develop a better diagnostic assay for HHV-8 infection, as there is no FDA-approved assay for this infection. Our initial studies have showed that a mixture of 4 antigens in the LIPS assay performs at least as well as an ELISA-based assay. In a subsequent study examining the sera of HIV+ and HIV- individual without Kaposi's sarcoma, but who are likely at higher risk for infection with HHV-8, the LIPS assay appears to be less sensitive than an ELISA in a non-Kaposi's population. These results have been recently submitted for publication. For EBV, we are examining the relationship between development of lymphoma and changes in antibody titers to EBV-specific proteins, as well as EBV viral load. Our data so far suggest that there is no relationship between anti-EBV antibody titers and development of lymphoma. We have recently completed determination of EBV content in purified B cells of patients with lymphoma as well as control patients, but similarly have not found a relationship between EBV load and subsequent development of lymphoma. Both HIV infection and antiretroviral nucleoside analogues (nucleoside reverse transcriptase inhibitors or NRTIs) are known to affect mitochondrial DNA content and mitochondrial function. A number of important clinical syndromes observed in HIV-infected persons relate to mitochondrial dysfunction including lactic acidosis, myopathy, cardiomyopathy, pancreatitis, peripheral neuropathy, and possibly lipodystrophy. Fatigue, one of the most prevalent complaints among persons with HIV infection, may also be the result of mitochondrial toxicity, though this has not been clearly established. Availability of minimally invasive tests to assess mitochondrial toxicity would greatly facilitate understanding of the contribution of mitochondrial dysfunction to clinical syndromes. Muscle and liver biopsies are currently considered to be the reference standards for the evaluation and diagnosis of mitochondrial toxicity in muscle and liver, but these invasive tests are impractical for routine and repeated evaluations. The recent development of a real-time polymerase chain reaction (PCR) assay to accurately quantify the mtDNA copy numbers per cell in peripheral blood mononuclear cells (PBMCs) may allow non-invasive assessment of mitochondrial toxicity. We have undertaken a pilot study to examine the relationship between fatigue and other clinical parameters and markers of mitochondrial dysfunction. We found no relationship between fatigue and mitochondrial content. We did find the mitochondrial content in adipose tissue, but not muscle or PBMCs, was significantly increased in untreated patients with HIV infection compared to uninfected controls, and significantly decreased in patients receiving antiretroviral therapy. In addition, by microarray we have identified a set of genes differentially expressed in Fat of HIV+ versus HIV- individuals. This study has been recently published. At present, there are no clear guidelines as to when antiretroviral (ARV) therapy for human immunodeficiency virus (HIV) should be stopped in the setting of elevated liver enzymes. In large part, this is due to a limited understanding of the natural history of ARV-related hepatotoxicity. We have undertaken a pilot study to estimate the prevalence of hepatic fibrosis in a cohort of HIV-infected patients who have chronically elevated transaminases while on ARV therapy in the absence of Hepatitis B (HBV) or C (HCV) coinfection. Liver biopsy specimens will be evaluated for fibrosis by microscopic examination,the current gold standard for assessing the nature and severity of liver disease. Fibrosis, as well as other histopathology, will be measured using a validated scoring system. To date, no patients have enrolled in the study or undergone liver biopsy. Significant liver abnormalities, primarily steatohepatitis, but also fibrosis, have been seen in the majority of patients. This study should provide clinically relevant information on the significance of elevated transaminases in HIV-infected patients without co-infection with HCV or HBV, and facilitate management of such patients. Biomarkers including D-dimer and IL-6 have been show to predict mortality in HIV-infected patients independent of CD4 and viral load. To better understand the mechanism leading to D-dimer elevation, we have undertaken a cross-sectional study to examine markers of coagulation, platelet function, endothelial activation and inflammation, and to identify correlates of elevated D-dimer levels. Our hope is that this analysis will provide insights into which of these pathways is leading to D-dimer elevation. To date we have enrolled approximately 230 HIV+ patients and HIV-volunteers. Preliminary analysis suggests that TNF-, sVCAM, and von Willebrand Factor correlate with levels of D-Dimer. These data suggest that ongoing monocyte activation plays a role in D-dimer elevation. Diagnosis of respiratory infections in HIV infected patients remain an expensive and laborious process. With funding from a Bench to Bedside award, we have undertaken a protocol to examine the utility of a resequencing microarray in the diagnosis of such infections. We plan to enroll 200 patients at the NIH and Washington Hospital Center. Our initial goal is to determine the sensitivity of the microarray chip compared to results obtained in the Microbiology Department. This protocol has enrolled 86 patients at the NIH to date and 10 at Washington Hospital Center. Customized chip development is proceeding in parallel, and we hope to be able to begin processing samples using the new developed chip by the end of the year.